Biochemistry 2004, 43, 13193-13203
13193
Identification of Ligand Binding Regions of the Saccharomyces cereVisiae R-Factor Pheromone Receptor by Photoaffinity Cross-Linking† Cagdas D. Son,‡ Hasmik Sargsyan,§ Fred Naider,§,| and Jeffrey M. Becker*,⊥ Department of Biochemistry, Cellular, and Molecular Biology, UniVersity of Tennessee, KnoxVille, Tennessee 37996-0845, Department of Chemistry, College of Staten Island and Institute for Macromolecular Assemblies, City UniVersity of New York, Staten Island, New York 10314, Ph.D. Program in Biochemistry and Chemistry, The Graduate School and UniVersity Center of The City UniVersity of New York, 365 5th AVenue, New York, New York 10016, and Department of Microbiology and UniVersity of Tennessee-Oak Ridge National Laboratory School of Genome Science and Technology, UniVersity of Tennessee, KnoxVille, Tennessee 37996-0845 ReceiVed February 11, 2004; ReVised Manuscript ReceiVed August 10, 2004
ABSTRACT: Analogues of R-factor, Saccharomyces cereVisiae tridecapeptide mating pheromone (H-TrpHis-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing p-benzoylphenylalanine (Bpa), a photoactivatable group, and biotin as a tag, were synthesized using solid-phase methodologies on a p-benzyloxybenzyl alcohol polystyrene resin. Bpa was inserted at positions 1, 3, 5, 8, and 13 of R-factor to generate a set of cross-linkable analogues spanning the pheromone. The biological activity (growth arrest assay) and binding affinities of all analogues for the R-factor receptor (Ste2p) were determined. Two of the analogues that were tested, Bpa1 and Bpa5, showed 3-4-fold lower affinity than the R-factor, whereas Bpa3 and Bpa13 had 7-12-fold lower affinities. Bpa8 competed poorly with [3H]-R-factor for Ste2p. All of the analogues tested except Bpa8 had detectable halos in the growth arrest assay, indicating that these analogues are R-factor agonists. Cross-linking studies demonstrated that [Bpa1]-R-factor, [Bpa3]R-factor, [Bpa5]-R-factor, and [Bpa13]-R-factor were cross-linked to Ste2p; the biotin tag on the pheromone was detected by a NeutrAvidin-HRP conjugate on Western blots. Digestion of Bpa1, Bpa3, and Bpa13 cross-linked receptors with chemical and enzymatic reagents suggested that the N-terminus of the pheromone interacts with a binding domain consisting of residues from the extracellular ends of TM5TM7 and portions of EL2 and EL3 close to these TMs and that there is a direct interaction between the position 13 side chain and a region of Ste2p (F55-R58) at the extracellular end of TM1. The results further define the sites of interaction between Ste2p and the R-factor, allowing refinement of a model for the pheromone bound to its receptor.
The yeast Saccharomyces cereVisiae is a sexual organism that manifests a conjugative response when opposite mating type cells, MATa and MATR, are mixed together (for reviews, see refs 1-5). The mating reaction is initiated by stimulation of cell surface receptors on each cell type by a polypeptide pheromone secreted from cells of the opposite mating type. Ste2p is a G protein-coupled receptor (GPCR)1 expressed on the surface of haploid cells of the MATa mating type, which is involved in this initiation event. Binding of the R-factor tridecapeptide mating pheromone (Trp-His-Trp-LeuGln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr), secreted by the MATR mating type, to Ste2p activates the pheromone signaling pathway by promoting dissociation of a coupled heterotrimeric G protein into its constituent R subunit and
βγ subunit complex on the cytoplasmic side of the plasma membrane. The activation of the receptor leads to a series of events, including G1 growth arrest, cellular elongation, gene induction, agglutinin biosynthesis, and ultimately cell fusion with the opposite MATR mating type cells. GPCRs constitute a major family of human proteins (6). To date, more than 1000 GPCRs have been identified, and these proteins recognize diverse signaling molecules, including neurotransmitters, sensory molecules, and chemotactic agents (7). The ubiquitous nature of GPCRs together with their highly specific ligand recognition makes them an important target for therapeutic agents (8, 9). Among the most important GPCR ligands are peptides, including hormones, growth factors, and pheromones. Understanding
† This work was supported by Grants GM22086 and GM22087 from the National Institutes of Health. F.N. holds the Leonard and Esther Kurtz Term Professorship at the College of Staten Island. * To whom correspondence should be addressed. E-mail: jbecker@ utk.edu. Phone: (865) 974-3006. Fax: (865) 974-4007. ‡ Department of Biochemistry, Cellular, and Molecular Biology, University of Tennessee. § City University of New York. | The Graduate School and University Center of The City University of New York. ⊥ University of Tennessee.
1 Abbreviations: BNPS-skatole, 2-(2′-nitrophenylsulfenyl)-3-methyl3′-bromoindolene; Bpa, 4-benzoyl-L-phenylalanine; BSA, bovine serum albumin; CNBr, cyanogen bromide; DIEA, N,N-diisopropylethylamine; ESI-MS, electron spray mass spectrometry; Fmoc, 9-fluorenylmethoxycarbonyl; GPCR, G protein-coupled receptor; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt, N-hydroxybenzotriazole; NA-HRP, NeutrAvidin-horseradish peroxidase conjugate; Nle, norleucine; OButt, tert-butyl; tBoc, tert-butoxycarbonyl; TAME, N-R-p-tosyl-L-arginine methyl ester; TFA, trifluoroacetic acid; Wang resin, (4-hydroxymethyl)phenoxymethyl on 1% cross-linked polystyrene resin (bead).
10.1021/bi0496889 CCC: $27.50 © 2004 American Chemical Society Published on Web 09/25/2004
13194 Biochemistry, Vol. 43, No. 41, 2004 the molecular mechanism of peptide recognition by their cognate GPCRs may lead to design of novel peptide analogues for treatment of disorders involving defective ligands and/or mutant receptors. While there has been considerable literature on the GPCR ligand binding sites for small molecules such as biogenic amines (10), relatively less information concerning the binding sites of peptide-responsive GPCRs exists. Photoreactive cross-linking studies, using 4-benzoyl-L-phenylalanine (Bpa)-containing peptide ligands, are a powerful complement to site-directed mutagenesis studies for mapping peptidereceptor interaction(s). Studies reported on parathyroid hormone (PTH), opioid receptor (ORL1) (11-17), substance P (18-20), cholecystokinin (21), and vasopressin (22) showed that Bpa-containing peptides can be successfully used for identification of receptor fragments that are interacting with photoactivatable peptide analogues. Recent work on human PTH receptor (23, 24), the secretin receptor (25), the ORL1 receptor (26), and the human angiotensin type I receptor (27) showed that specific contact residues can be mapped after photoaffinity labeling by sequencing of the cross-linked fragment and site-directed mutagenesis in the region of cross-linking. A new method called the methionine proximity assay took advantage of modified analogues of Bpa, e.g., p,p′-nitrobenzoylphenylalanine (NO2Bpa), that exhibit selectivity for methionine residues (28). This unique property allowed the identification of a contact site by methionine scanning of the putative contact residues of three GPCRs, followed by analysis of the cross-linked receptor. All of these studies using Bpa analogues revealed that generation of covalently linked ligand-receptor conjugates has become a valuable tool for the identification of the crosslinked domains, allowing mapping of the interface between a peptide ligand and its GPCR (29). Previously, we reported this general approach to identifying directly the interaction between position 1 of the R-factor and a region between residues 251 and 294 of the receptor Ste2p (30). However, that study was limited to the use of only one Bpa-bound R-factor analogue. Here we report the evaluation of a series of photoactivatable analogues containing Bpa at various positions throughout the R-factor pheromone. An important aspect of this study is the use of biotin as a tag allowing detection of cross-linking using avidinbased horseradish peroxidase (HRP) conjugates and eliminating problems previously encountered with radioactive iodine (30). Four analogues were selectively cross-linked into the R-factor binding site of Ste2p. Chemical and enzymatic fragmentation of the cross-linked receptor allowed us to identify contacts between the position 3 and 13 side chains of the pheromone and Ste2p residues in the EL2-TM5 and/ or TM6-EL3-TM7 region and first transmembrane domain, respectively. EXPERIMENTAL PROCEDURES Organisms. S. cereVisiae DK102 [MATa ste2::HIS3 bar1 leu2 ura3 lys2 ade2 his3 trp1] transformed with pNED1[STE2] (31) was used in binding studies and in the growth arrest assays of various R-factor analogues. Strain BJ2168 was used as a parental strain to construct BJS21 by deleting STE2 using the kanamycin deletion cassette according to a standard protocol (32). The BJS21 [MATa ste2::KanR, prc1-
Son et al. 407, prb 1-1122, pep4-3, Leu2, trp1, ura 3-52] strain transformed with pNED1[STE2] was used in cross-linking studies. All strains exhibited a similar binding of the R-factor. However, the BJS21 pNED strain lacked several peptidases, so it was used to increase the yield of Ste2p in experiments involving cross-linking. Chemical Reagents. All reagents and solvents used for the solid-phase peptide synthesis of the photoactivatable peptides were analytical grade and were purchased from Advanced ChemTech (Louisville, KY), VWR Scientific (Piscataway, NJ), or Aldrich Chemical Co. (Milwaukee, WI). Highperformance liquid chromatography (HPLC) grade dichloromethane (CH2Cl2), acetonitrile (ACN), methanol (MeOH), and water were purchased from VWR Scientific and Fisher Scientific (Springfield, NJ). Synthesis of N-R-Fmoc-Protected R-Factor Analogues. N-R-Fmoc-protected R-factor analogues needed for the preparation of their biotinylated derivatives were synthesized by solid-phase peptide synthesis on an Applied Biosystems 433A peptide synthesizer (Applied Biosystems, Foster City, CA) starting with N-R-Fmoc-Tyr(OBut)- or N-R-Fmoc-BpaWang resin (0.7 mmol/g substitution, Advanced ChemTech). In all of the peptides, L-norleucine (Nle), which is isosteric with L-methionine, was incorporated at position 12 to replace the naturally occurring L-methionine to prevent oxidation of the sulfur atom of this amino acid during peptide synthesis and purification. Replacement of Met with Nle was shown previously to result in an analogue with activity and receptor affinity equal to those of the native pheromone (33). Since all analogues have Nle in place of Met12, this residue is eliminated from the abbreviated names for simplicity. The 0.1 mmol FastMoc chemistry of Applied Biosystems was used for the elongation of the peptide chain with an HBTU-, HOBt-, and DIEA-catalyzed, single coupling step using 10 equiv of protected amino acids for 30 min. Upon completion of chain assembly, the N-R-Fmoc group was not removed from the peptide chain. CleaVage of the Peptide. The N-R-Fmoc-protected peptidyl resin was washed thoroughly with 1-methyl-2-pyrrolidinone and dichloromethane and dried in a vacuum for 2 h. The peptide was cleaved from the resin support with simultaneous side chain deprotection using a cleavage cocktail containing trifluoroacetic acid (10 mL), crystalline phenol (0.75 g), thioanisole (0.5 mL), and water (0.5 mL) at room temperature for 1.5 h with the omission of 1,2-ethanedithiol from the cleavage reaction, because it was known to transform Bpacontaining peptides to cyclic dithioketal derivatives (34). The filtrates from the cleavage reaction were collected, combined with trifluoroacetic acid washes of the resin, concentrated under reduced pressure, and treated with cold ether to precipitate the crude product. Purification and Characterization. The crude peptide so obtained was purified by reversed-phase HPLC (HewlettPackard Series 1050) on a semipreparative Waters µ-Bondapack C18 (19 mm × 300 mm) column with detection at 220 nm. The crude product (50 mg) was dissolved in ∼4 mL of aqueous acetonitrile (20%) containing 0.025% TFA, applied to the column, and eluted with a water/acetonitrile linear gradient containing 0.025% TFA (0 to 70% acetonitrile over 2 h at a flow rate of 5 mL/min). The fractions were collected and analyzed at 220 nm by reversed-phase HPLC (HewlettPackard Series 1050) on an analytical Waters µ-Bondapack
R-Factor Binding Sites
Biochemistry, Vol. 43, No. 41, 2004 13195
Table 1: Βpax,Lys7(biotinylamidocaproate)R-Factor Analogues, Yields of Biotinylation, and Results of Mass Spectral Analysis yield of biotinylated product (%) 1
7
12
13
1
Bpa ,K (biotinylamidocaproate),Nle ,Y -R-factor (Bpa ) Bpa3,K7(biotinylamidocaproate),Nle12,Y13-R-factor (Bpa3) Bpa5,K7(biotinylamidocaproate),Nle12,Y13-R-factor (Bpa5) Bpa8,K7(biotinylamidocaproate),Nle12,Y13-R-factor (Bpa8) Bpa13,K7(biotinylamidocaproate),Nle12-R-factor (Bpa13)
51.0 89.9 90.2 86.2 84.1
c
purity (%) by HPLC
E280a
MW (calculated)
ESI-MSb
>99 >99 >99 >99 >99
14 000 14 000 19 500 19 500 18 000
2070 2070 2128 2159 2093
2070.2 2070.2 2127.7 2159.0 2093.0
a The extinction coefficients were calculated with DNASTAR (Lasergene, Madison, WI) on the basis of the peptide sequence. b Mass spectral analysis of biotinylated analogues was performed by C. Soll (Hunter College, City University of New York). c This synthesis was not optimized.
C18 (3.9 mm × 300 mm) column. Fractions that were more than 99% homogeneous were pooled and subjected to lyophilization. The purity of the final peptide was assessed by analytical HPLC using two different solvent systems (10 to 55% acetonitrile gradient, 15 min, with 0.025% trifluoroacetic acid, and 50 to 80% methanol gradient, 30 min, with 0.025% trifluoroacetic acid) and ESI-MS (Hunter College, City University of New York, New York, NY). Biotinylation of N-R-Fmoc-Protected R-Factor Analogues. Biotinylation of N-R-Fmoc-protected R-factor analogues was carried out using biotinylamidocaproate N-hydroxysuccinimide ester (Sigma). The N-R-Fmoc-protected R-factor analogue (10.0 mg, 5 µmol) was dissolved in 0.5 mL of cold (0 °C) DMF, and then the same volume (0.5 mL) of cold buffer (Na2B4O7, pH 9.5, 50 mM) was added. The solution was stirred at 0 °C (ice bath) for 2 min, and 10.0 mg (20 µmol) of biotinylamidocaproate N-hydroxysuccinimide ester (Sigma) dissolved in 0.5 mL of cold DMF (0 °C) was added very slowly. The reaction was monitored by analytical reversed-phase HPLC using a linear gradient of 20 to 70% ACN in 30 min, and when the starting peptide disappeared (20-40 min) 0.5 mL of a 20% solution of piperidine in DMF was added and the reaction mixture was stirred at room temperature for 30 min until the Fmoc group was fully deprotected, as monitored by analytical HPLC. The reaction mixture was neutralized with 0.2 M HCl, and injected immediately onto a preparative HPLC column (conditions for preparative HPLC, 20 to 70% ACN over 150 min; column, Waters µ-Bondopack C18, 19 mm × 300 mm). The main fraction was collected and lyophilized. The purity of the biotinylated peptide was >99% as determined by analytical HPLC, and the molecular weight of peptide was confirmed by mass spectral analysis. The overall recovery of the purified biotinylated peptide was 85-90%. Growth Arrest (Halo) Assay. Solid MLT medium [6.7 g/L yeast nitrogen base without amino acids (Difco), 10 g/L casamino acids (Difco), 20 g/L glucose, 0.058 g/L adenine sulfate, 0.026 g/L arginine, 0.058 g/L asparagine, 0.14 g/L aspartic acid, 0.14 g/L glutamic acid, 0.028 g/L histidine, 0.058 g/L isoleucine, 0.083 g/L leucine, 0.042 g/L lysine, 0.028 g/L methionine, 0.69 g/L phenylalanine, 0.52 g/L serine, 0.28 g/L threonine, 0.042 g/L tyrosine, 0.21 g/L valine, and 0.028 g/L uracil] (35) was overlaid with 4 mL of a S. cereVisiae DK102pNED cell suspension (2.5 × 105 cells/mL of Nobel agar). Filter disks (sterile blanks from Difco), 8 mm in diameter, were impregnated with 10 µL portions of peptide solutions at various concentrations and placed onto the overlay. The plates were incubated at 30 °C for 24-36 h and then observed for clear zones (halos) around the disks. The data were expressed as the diameter of the
halo, which includes the diameter of the disk. A minimum value for growth arrest is 9 mm, which represents the disk diameter (8 mm) and a small zone of inhibition. All assays were carried out at least three times with no more than a 2 mm variation in halo size at a particular amount applied for each peptide. The reported values represent the mean of these tests. Binding Competition Assay with [3H]-R-Factor. This assay was performed using strain DK102pNED and tritiated R-factor prepared by reduction of [dehydroproline8,Nle12]R-factor as described previously (33). In general, cells were grown at 30 °C overnight and harvested at a density of 1 × 107 cells/mL by centrifugation at 5000g and 4 °C. The pelleted cells were washed twice in ice-cold buffer [PPBi, 0.5 M potassium phosphate (pH 6.24) containing 10 mM TAME, 10 mM sodium azide, 10 mM potassium fluoride, and 1% BSA (fraction IV)] and resuspended at a density of 4 × 107 cells/mL. The binding assay was started by addition of [3H]-R-factor (6 nM) and various concentrations of nonlabeled peptide (140 µL) to a 560 µL cell suspension. Analogue concentrations were adjusted using UV absorption at 280 nm and the corresponding extinction coefficients (Table 1). After incubation for 30 min, triplicate samples of 200 µL were filtered and washed over glass fiber filtermats using the Standard Cell Harvester (Skatron Instruments, Sterling, VA) and placed in scintillation vials for counting. Each experiment was carried out at least three times with similar results in each assay. The Ki values were calculated by using the equation of Cheng and Prusoff, where Ki ) IC50/(1 + [ligand]/Km) (36). Cross-Linking of R-Factor Analogues to Ste2p. BJS21 and BJS21 pNED membranes (220 µg/mL of total protein) (31) were incubated with 975 µL of PPBi buffer (with 0.1% BSA) in siliconized microfuge tubes for 10 min at ambient temperature. Bpa-scanned biotinylated R-factor analogues (10 nM Bpa1, Bpa3, or Bpa13 and 20 nM Bpa5 or Bpa8) were added, and the reaction mixture was incubated for 30 min at room temperature with gentle mixing. The reaction mixture was aliquoted into three wells of a chilled 24-well plastic culture plate preblocked with PPBi (0.1% BSA). Division of the reaction mixtures into separate wells kept the depth of the samples minimal for efficient UV penetration of the sample. The samples were held at 4 °C and irradiated without the culture plate lid at 365 nm for 45 min in the case of Bpa1, Bpa3, or Bpa13 and for 90 min for Bpa5 or Bpa8 at a distance of 12 cm in a Stratlinker (Stratagene, La Jolla, CA). Membrane samples were recombined in siliconized microfuge tubes and washed twice by centrifugation (14000g) with PPBi (0.1% BSA). Membrane pellets were dissolved in 5 µL of sample buffer [0.25 M Tris-HCl (pH 8.8), 0.005%
13196 Biochemistry, Vol. 43, No. 41, 2004 bromophenol blue, 5% glycerol, 1.25% β-mercaptoethanol, and 2% SDS]. Samples were heated to 37 °C for 10 min and separated by SDS-PAGE (10% gel, 30 mA). In competition cross-linking experiments, 100× cold R-factor was added together with biotinylated peptide. For subsequent receptor digestion analysis, the band between 45 and 60 kDa covering the cross-linked Ste2p mass was excised and placed into dialysis tubing (molecular weight cutoff of 30 000) with buffer [0.2 M Tris-acetate (pH 7.4), 1.0% SDS, and 100 mM dithiothreitol]. Electroelution was performed by placing dialysis tubing that contains the gel in a horizontal electrophoresis chamber with running buffer [50 mM Tris-acetate (pH 7.4), 0.1% SDS, and 0.5 mM sodium thioglycolate]. Elution was carried out at 100 V for 3 h. The buffer in the dialysis tubing was transferred to a Millipore Ultrafree-15 centrifuge filter device (molecular weight cutoff of 30 000) and concentrated by centrifugation. The concentrated sample was washed three times with 50 mM Tris-HCl (pH 7.5). The cross-linked samples were resolved with SDS-PAGE, transferred to a PVDF membrane, and assayed for the detection of proteins that are covalently linked with biotinylated analogues with the NeutrAvidinHRP conjugate (NA-HRP) (Pierce). All the gels used in these blots were analyzed by staining with coomassie blue to ensure efficient transfer of the protein to the membrane. Digestion of Cross-Linked Ste2p. Cross-linked samples were digested with CNBr, trypsin, or BNPS-skatole. For CNBr digestion, samples were dissolved in 70% formic acid, and 200 mg/mL CNBr was added. For trypsin digestion, samples were dissolved in trypsin digestion buffer [100 mM Tris-HCl (pH 8.5)], and 6.25 µg of trypsin (sequencing grade modified trypsin, Roche) was added to 250 µg of total proteins; after incubation for 6 h, a second batch of trypsin (6.25 µg) is added to achieve complete digestion. Ste2p contains a lysine residue (K269) followed by a proline residue (P270) in extracellular loop 3, which makes it less likely to be digested by trypsin. Nevertheless,we found that this region is reproducibly susceptible to trypsin digestion under the conditions described above. For BNPS-skatole digestion, samples were dissolved in 50% acetic acid with addition of 10 mg/mL BNPS-skatole. To prevent anomalous cleavage of Ste2p at histidine and tyrosine residues, 100fold molar excess of tyrosine was added to the reaction mixture during BNPS-skatole digestion (37). Deglycosylated samples for BNPS-skatole digestion were prepared by treatment with PNGase F (500 units/µL, BioLabs). Briefly, ∼10 µL of partially purified Ste2p-containing membrane proteins (1 µg/µL) was incubated with 5 µL of 10× glycoprotein denaturing buffer (5% SDS and 10% β-mercaptoethanol) at 100 °C for 10 min. After 10 min, 5 µL of 10× G7 buffer [0.5 M sodium phosphate (pH 7.5)] and 5 µL of 10% NP-40 were added. Finally, 5 µL of PNGase F and 20 µL of water were added (total volume, 50 µL), and the mixture was incubated at room temperature overnight. All digestion reactions took place under nitrogen and in complete darkness at 37 °C. After digestion, samples were dried by vacuum centrifugation, resuspended in 50 µL of 0.5 M Tris (pH 8.25), and re-dried. The digested samples were dissolved in loading buffer (Bio-Rad) and resolved on 12% NuPAGE Bis-tris gels with MES running buffer (Invitrogen).
Son et al. RESULTS Synthesis of R-Factor Analogues. N-R-Fmoc-protected R-factor analogues containing Bpa1, Bpa3, Bpa5, Bpa8, or Bpa13 were prepared by automated solid-phase synthesis using standard coupling and deprotection protocols. During several of the syntheses, we noted partial Fmoc removal occurred using the “complete wash protocol” of the Applied Biosystems 433A synthesizer. This problem was eliminated by washing with only DCM. N-R-Fmoc-protected Bpacontaining R-factor analogues were purified by semipreparative HPLC (Waters µ-Bondopack C18, 125 Å, 19 mm × 300 mm, gradient from 20 to 60% acetonitrile over 150 min). After lyophilization, they were used in the synthesis of the corresponding biotinyl-amidocaproate (BioACA) derivatives. Thus, all biotinylated analogues used in this study were biotinylated at the -amine of Lys7, and a spacer was used to ensure that the biotin group was available to the avidin detection protein. The final products containing both Bpa and biotin were more than 99% pure as determined by analytical reversed-phase HPLC and had the expected molecular weight (Table 1). Receptor Affinities of Bpa-Scanned Biotinylated R-Factor Analogues. To achieve efficient cross-linking of a ligand to its receptor, it is necessary to employ ligand analogues that bind tightly. Previous studies showed that Bpa replacements were tolerated well at positions 1 (Trp), 3 (Trp), 5 (Gln), 7 (Lys), 8 (Pro), 12 (Met), and 13 (Tyr) of R-factor pheromone, with Kd values indicating 4-45-fold poorer binding in comparison to that of R-factor (30). On the basis of these data and our current working model for pheromone binding to receptor (38), we chose photo-cross-linkable analogues with Bpa at positions 1, 3, 5, 8, and 13 to determine points of contact between the peptide and its receptor. This placed potential cross-link sites in side chains throughout the pheromone. The position 7 side chain was used for the biotin tag for detection purposes (39). Binding affinities of the Bpa-scanned biotinylated R-factor analogues were determined by assessing their ability to compete with [3H]-R-factor as described in Experimental Procedures. Analogues containing Bpa1 and Bpa5 exhibited an only 3-4-fold reduction in binding affinity compared to that of the native R-factor (Figure 1 and Table 2). The binding affinity for Bpa13 was relatively poor (∼12-fold lower than that of the wild type). [Bpa8]-R-Factor showed very poor competition and did not compete fully at the highest tested concentration, making the binding affinity for this analogue only an estimate. [Bpa3]-R-Factor exhibited a comparatively intermediate binding affinity of ∼40 nM, although this is also only an estimate as full competition did not occur at the highest tested concentration. BioactiVities of Bpa-Scanned Biotinylated R-Factor Analogues. For the cross-linking studies to relate to a biologically relevant receptor-ligand interaction, it is important to determine whether the ligand has biological activity. Therefore, we investigated the ability of the Bpa/biotinylated R-factor analogues of this study to cause growth arrest of MATa cells. Semilogarithmic plots of halo diameter versus the amount of peptide applied to the disk were all linear and exhibited similar slopes (data not shown). All analogues used in this assay were stable to enzymatic cleavage under the conditions that were tested as strains used in this analysis
R-Factor Binding Sites
Biochemistry, Vol. 43, No. 41, 2004 13197
FIGURE 2: Western blot analysis of BJS21 pNED (expresses Ste2p) and BJS21 (∆Ste2p) photo-cross-linked membranes. Membranes photo-cross-linked with various Bpa-containing R-factor analogues were dissolved in Tricine sample buffer and resolved with a SDSPAGE 10% gel. Proteins were transferred to a PVDF membrane and probed with NA-HRP to detect the biotin signal on the analogue.
FIGURE 1: Competition binding assay of various R-factor analogues vs tritiated R-factor. Competitors were cold R-factor (9), Bpa1 (2), Bpa3 (1), Bpa5 ([), Bpa8 (b), and Bpa13 (0). Table 2: Summary of Receptor Affinities and Biological Activities for Bpa-Scanned Biotinylated R-Factor Analogues wild type Bpa1 Ki (nM) biological activitya % affinity % activity
6 0.4 100 100
20 1.5 30 23
Bpa3
Bpa5
Bpa8
Bpa13
∼40 3.5 15 10
25 9.4 24 4
∼160 ∼50b ∼4
